TGF-β1和(或)三氧化二砷作用于HL-60细胞后P27^(Kip1)表达及凋亡情况的研究

Levels of P27^(Kip1) Expression and Apoptosis in HL-60 Cells after Treatment with TGF-β1 and/or Arsenic Trioxide

本研究观察三氧化二砷(As2O3)和/或TGF-β1对HL-60细胞凋亡及细胞周期的影响,以及作用前后P27Kip1、内源性TGF-β1、cyclinE和BCL-2的变化情况。As2O3和/或TGF-β1作用于HL-60细胞,用细胞形态学观察及流式细胞术检测细胞凋亡和细胞周期改变情况,应用免疫组织化学检测药物处理前后P27Kip1、内源性TGF-β1、cyclinE以及BCL-2表达的变化情况。结果表明:As2O3和TGF-β1单用均可以诱导HL-60细胞发生凋亡,联合处理对HL-60细胞生长抑制作用强于单药处理,其中5μmol/LAs2O3作用最强,5ng/mlTGF-β1处理可引起HL-60细胞的细胞周期阻滞于G1期(p<0.05)。5ng/mlTGF-β1和联合处理组P27Kip1的表达较对照组增强(p<0.05);5μmol/LAs2O3处理组P27Kip1的表达与对照组比较无明显差异(p>0.05)。5ng/mlTGF-β1和联合处理组均可见cyclinE蛋白表达下调(p<0.05)。5μmol/LAs2O3和联合处理组可见内源性TGF-β1表达上调(p<0.05)。结论:As2O3、外源性TGF-β1均可诱导HL-60细胞凋亡,联合处理组诱导凋亡作用强于对照组和单药处理组,TGF-β1处理可引起HL-60细胞周期阻滞于G1期。TGF-β1诱导HL-60细胞凋亡过程中P27Kip1表达增强,拮抗cyclinE和BCL-2的作用,细胞周期阻滞,诱导细胞发生凋亡,这表明P27Kip1是TGF-β引起细胞周期阻滞的关键效应物,外源性TGF-β1又通过上调内源性TGF-β1的表达增强As2O3诱导原代APL细胞凋亡的作用。

This study was purposed to investigate the effects of TGF-β1 and arsenic trioxide （As20s ） on cell apoptosis, cell cycle and changes of P27Kip1 , endogenous TGF-β1, cyclin E and BCL-2 in HL-60 cells, and to explore the relationship between expression of P27Kip1 and apoptosis induced by As2O3 and/or TGFβ1. Cell apoptosis and cell cycle changes of HL-60 cells treated with As2 O3 and/or TGFβ1 were detected by cytomorphologic observation and flow cytometry, the protein expressions of P27^Kip1 ,TGF-β1 ,cyclin E and BCL-2 were measured by immunohistochemistry. The results showed the effect of 5 μmot/L of As2O3 was the most strong among the different concentration of As2O3, and the effect on apoptosis at 48 hour was more strong than that at 24 hours （ p 〈 0.05 ）. The TGF-β1 （ 5 ng/ml ） induced arrest of cells in G1 phase （p 〈 0.05 ） compared with As2O3 alone and As2O3 combined with TGF-β1, while there was no difference with control. P27^Kip1 expression was up regulated （p 〈 0.05 ）, cyclin E and BCL-2 expression was down-regulated （p 〈 0.05 ） in TGF β1-treated group. BC1-2 expression was down regulated, endogenesis TGFβ1 expression was up regulated （p 〈0.05 ）, and the level of P27^Kipl and cyclin E were not changed in As2O3-treated group （p 〉0.05）. The down-regulating effect of TGF-β1 combined with As2O3 on BCL-2 protein was more strong than that in single factor treated group （p 〈 0.05 ）. It is concluded that TGFβ1 induces cell cycle arrest and apoptosis in HL-60 cells, while the P27klpl expression is up regulated. P27 protein is the key effector of TGF[3-induced cell cycle arrest. The effect of TGF-β1 combined with As2O3 on apoptosis as well as the down-regulation of BCL-2 protein in HL-60 cells is more strong than that in single factor-treated groups, that indicates the passages linking up each other.